Human prion diseases are a diverse group of neurodegenerative disorders that include Creutzfeldt-Jakob disease (CJD), fatal insomnia (familial and sporadic), Gerstmann-Straussler-Scheinker (GSS) disease and variably protease-sensitive prionopathy (VPSPr). Despite large phenotypic variability, a common denominator of all these diseases is the accumulation of proteinase-resistant aggregates of prion protein, PrPSc. While some of these diseases (e.g., CJD) are known to be highly transmissible, transmissibility potential of other members of this group is less well established. The long-term objective of this research is to advance the understanding of the molecular basis, transmissibility properties and phenotypic variability of these disorders. Three interrelated specific aims are proposed. Specific Aim 1 focuses on transmissibility properties of GSS subtypes and biochemical and conformational features of PrPSc that determine phenotypic variability of these diseases. This aim includes experiments with PrPSc from GSS brain tissue as well as novel synthetic prions, the transmissibility of which will be studied using new lines of transgenic mice expressing human prion protein. Specific Aim 2 examines the role of PrP glycosylation in strain characteristics, phenotype determination, and transmissibility. Preliminary data indicate that, in contrast to observations on mouse prions, glycan-free human prions replicate more efficiently but reduce phenotypic differences. This research relies on a recently generated line of transgenic mice expressing unglycosylated human PrP. Specific Aim 3 seeks to elucidate the molecular basis of the recently discovered variably protease sensitive prionopathy (VPSPr). Specific features have been preliminarily identified in PrPSc that point to distinct conformational characteristics associated with this disease and a possible pathogenic mechanism. These PrPSc characteristics will be further examined using a battery of biochemical and conformational assays. Furthermore, new data suggest that, although VPSPr transmits poorly to transgenic mice expressing normally glycosylated human prion protein, transmission of this disease to transgenic mice expressing unglycosylated human PrP might be much more efficient. These transmissibility properties will be further assessed and characterized.